J Life Sci Res 2018; 16: 1-7© 2018 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Original

Characterization of an Angiotensin I-Converting Enzyme Inhibitor Substance inTomato Juice
Momoko IMAI † 1, 2, Hiroshi INUI1, Hitoshi ASHIDA3, Kimio NISHIMURA4, Takahiro INAKUMA5, Nobuhide FUJITAKE3
1

Department Nutrition, Osaka Prefecture University, Habikino 3-7-3, Habikino, Osaka 583-8555, Japan

2

Faculy of Human Development, Soai University, Nankounaka 4-4-1, Suminoeku , Osaka, Osaka 559-0033, Japan

3

Graduate School of Agricultural Science, Faculty of Agriculture, Kobe University,

Rokkoudaicho 1-1, Nadaku, Kobe, Hyogo 657-8501, Japan
4

Faculty of Human Life Sciences, Doshisha Women’s College of Liberal Arts,

Genbucho 602, Imadegawa Dori Teramachi Nishi-iru, Kamigyouku, Kyoto, Kyoto 603-0893, Japan
5

Faculty of Health Science, Kyoto Koka Women's University, Nishikyogoku Kadonocho 38, Ukyoku, Kyoto, Kyoto 615-0882, Japan

Received 10 October 2018; accepted 20 November 2018
Abbreviations: ACE, angiotensin I-converting enzyme; COSY, correlation spectroscopy; HMQC, heteronuclear
multiple quantum correlation; HPLC, high-performance liquid chromatography; HPSEC, high pressure size exclusion
chromatography; MP, mobile phase; MW, molecular weight; NMR, nuclear magnetic resonance; poly-GalA,
polygalacturonic acid; RI, refractive index; RT, retention time
Key words: tomato juice, angiotensin I-converting enzyme, inhibitory activity, uronic acid, oligosaccharide, nuclear magnetic resonance

1 Abstract

in the world in the age group of 25 years or older are likely toThis study was performed to purify a substance with

contract hypertension2. One of the main mechanisms leading toangiotensin I-converting enzyme (ACE) inhibitory activity fromhypertension is the rennin-angiotensin system. In this system,

tomato juice. Tomato juice was centrifuged, and the supernatantthe angiotensin I-converting enzyme (ACE) converts angiotensin

was filtered through a membrane filter and applied to a cation

I to angiotensin II, which is a strong vasopressor substance andexchange, hydrophobic and reverse phase chromatography,

plays a crucial physiological role in blood-pressure regulation3.

sequentially. The impermeable fractions with molecular weights

This mechanism suggests that the inhibition of ACE is a usefulof about 1.0 kDa were collected. The fraction was further

therapeutic approach for hypertension remedy. It has been also

purified by high-performance liquid chromatography. Then, theshown that the ACE inhibitor dosage restraints hypertensive

fractions containing ACE inhibitory activity were concentrated

onset in spontaneously hypertensive rats4, Several ACE inhibitor

using centrifugal ultrafiltration and lyophilized. The lyophilized

synthetic drugs are used for treatment of high blood pressure.

sample was subjected to nuclear magnetic resonance analysis.

However, several side effects of these medicines were reported:

The results of the analyses suggested that the fraction withcough, taste abnormality, skin rash, and angioneurotic edema5.

ACE inhibitory activity from tomato juice contains a structure

Therefore, extensive search of safe, innovative substances,

containing less than 10 sugar moieties of uronic acid ofshowing effective ACE inhibitory activity is carried out using

approximately 3.0 kDa.

various natural materials, including food.
It has been reported that some foods possess ACE
inhibitory properties: herbs and phaseolus coccineus6, black

2 Introduction
It is known that cerebral vascular disease and ischemic heartmatpe 7 , chenopodium quinoa 8 , morinda citrifolia 9 , anddisease are two out of four major causes of death in Japanesemushroom10. Furthermore, specific substances in foods, such as

1people, and high blood pressure is a major risk factor for them .

polyphenol in tea11, peptides from sardine meat protein12, animal

World Health Organization reported that one out of three people

protein13-15, oligopeptides in porphyra yezoensis16, nicotianamine
in various beans 17, anthocyanins from hibiscus sabdariffa 18,†

Corresponding author, E-mail: imai@soai.ac.jp

chitooligosaccharide derivatives and phlorotannins from marine

2resources19, and vegetables20, also show ACE inhibitory effects.

Devices, Pall, NY, USA) at 5,000 rpm for 20 min, the inhibitory

Tomato juice is a popular beverage among the health-activity was confirmed in the impermeable fraction with aconscious people in Japan. Spontaneously Hypertensive

molecular weight (MW) over 1.0 kDa, which was collected andRats(SHR) showed significant reduction in blood pressurelyophilized.

on ingestion of tomato juice 21 . Therefore, we undertook

Step 3: The freeze-dried sample was dissolved in 0.05%

the purification and structure determination of the fraction

trifluoroacetic acid (TFA) and further purified using highresponsible for ACE inhibitory activity from tomato juice.

performance liquid chromatography (HPLC) system (Waters,
Massachusetts, USA) equipped with pumps with controller

3 Materials and Methods(600, Waters), detector UV/VIS (2487 Dualλ Absorbance3.1 Samples and reagents

Detector, Waters), differential refractometer (2410 Refractive

Tomato juice products (salt-free and extracted juice 100%)

Index Detector, refractive index (RI) detector, Waters), degasser

were purchased from KAGOME (Tokyo, Japan) products. All(ERC-3415α, ERC, Saitama, Japan), autosampler (717 plus

other chemicals were of reagent grade and were obtained from

Autosampler, Waters), Cosmosil 5C18-PAQ (id. 20 mm × 250

Nacalai Tesque (Kyoto, Japan).

mm, Nacalai Tesque). The eluate was detected at 280 nm. The

3.2 Purified procedure of ACE inhibitor substance from Tomato

mobile phase (MP) of HPLC consisted of 0.05% TFA. The

juicecolumn was operated at temperature at 25 °C, the flow rate was

The purification procedure is shown in Fig. 1. The steps

3.0 mL/min, and the injection volume was 2.0 mL. The HPLC

were as follows:

system was connected to a fraction collector (SF-2120, Toyo
Roshi), which was programmed to collect each 1.5 mL/min, andfractions containing ACE inhibitory activity were collected. The
fraction was subjected to centrifugal ultrafiltration to remove
TFA, and the impermeable fraction (MWCO > 1.0 kDa) was
freeze-dried.
Step 4: The freeze-dried sample was dissolved in ultrapure
water, and purified by HPLC using a Shodex-Ohpak SB-2002.5
column (id. 20 mm × 300 mm, Showa Denko, Tokyo, Japan) andthe eluate was monitored at 450 nm. The MP of HPLC consistedof ultrapure water, the flow rate was 3.0 mL/min, and injection
volume was 2.0 mL. The fractions with ACE inhibitory activity
were collected, and the impermeable fraction (MW> 1.0 kDa)
was freeze-dried.
Fig.1

Process of purification.

Step 5: The freeze-dried sample in Step 4 was dissolved in
5.0% acetonitrile solution with 0.1% TFA, and purified by HPLC
as described in Step 4, substituting MP with 5.0% acetonitrile

Step. 1: Tomato juice (7.2 L) was centrifuged at 8,000

solution containing 0.1% TFA, and the eluate was monitored at

× g for 30 min at 4°C, and the obtained supernatant was

280 nm. The flow rate was 3.0 mL/min, and injection volume

centrifuged at 11,000 × g for 60 min at 4°C. The supernatant was

was 2.0 mL.

concentrated onto a 0.45 μm-pore size membrane filter (293 mm

The fractions with ACE inhibitory activity were collected

diameter, ADVANTEC CELLULOSE ACETATE, Toyo Roshi,

and subjected to centrifugal ultrafiltration to remove TFA, andTokyo, Japan) under vacuum.

the impermeable fraction (MWCO> 1.0 kDa) was freeze-dried.

Step 2: The filtrate was separated on a cation exchange

Step 6: The freeze-dried sample was dissolved in 0.1 M

resin (DOWEX 50W-X2, Wako Pure Chemicals, Chiba, Japan)

sodium nitrate solution, and purified by HPLC as described inusing an open glass column (id. 40 mm × 300 mm), and theStep 4, substituting MP with 0.1 M sodium nitrate solution, andflow-through was collected. The solution was applied to a

the eluate detected at 410 nm. The flow rate was 1.5 mL/min,column packing with hydrophobic resin (Supelite DAX-8,

and the injection volume was 2.0 mL. The fractions with ACE

Sigma-Aldrich; id. 40 mm × 300 mm), and the flow-throughinhibitory activity were collected and freeze-dried and used as

was recovered again. The solution was applied to a column

the purified sample.

packing with reversed phase silica gel (Cosmosil 75 C18-OPN,

3.3 Determination of the ACE inhibitory activity

Nacalai Tesque; id. 40 mm × 300 mm), and the flow-throughACE inhibitory activity was measured by the enzyme

was obtained. Using centrifugal ultrafiltration (Molecular Weight

method (ACE kit-WST, Dojindo Molecular Technologies, Inc.),

Cut-Off (MWCO) <1.0 kilodalton (kDa), Macrosep Centrifugal

which measured the amount of 3-hyroxybutylic acid generated

3

from 3-hydroxybutylyl-Gly-Gly-Gly, and expressed as an IC50

System 500 (Agilent Technologies, California, USA). 1 Hvalue.

spectroscopy was acquired under following conditions: delay of

3.4 Measurement of molecular weight using high-pressure size

1.5 s, acquisition time of 3.5 s, flip angle of 45°, and 128 scans.

exclusion chromatography (HPSEC)

13

C spectroscopy was acquired under following conditions: delay

The purified sample was solubilized in 0.1 M sodiumof 1.5 s, acquisition time of 1.5 s, flip angle of 45°, and 16,834

nitrate solution, and its molecular weight was measured using

scans. Correlation spectroscopy (COSY) was acquired withHPSEC. The HPLC system was described in Step 3. MP was

1.0 s delay, acquisition time of 0.22 s, flip angle of 90°, and 16

0.1 M sodium nitrate, using polyethylene glycol (MW: 300, 400,

scans. Heteronuclear multiple quantum correlation (HMQC) was

600, 1,000, 1,500, 2,000, 3,350, 4,000, 8,000, Sigma-Aldrich) as

acquired with 0.13 s delay, acquisition time of 1.0 s, flip angle ofstandard substance. Analysis of gel permeation chromatography90°, 32 scans, and heteronuclear multiple bond coherence waswas performed on a millennium 32-J software (Showa Denko),acquired with 1.0 s delay, acquisition time of 0.13 s, couplingwith a Shodex-Ohpak SB-803HQ column (id. 20 mm × 300 mm,

constant 8 Hz, flip angle of 90°, and 128 scans.

Showa Denko) and Shodex-Ohpak SB-G guard column (id. 6.0mm × 50 mm, Showa Denko). The exclusion limit molecular

4 Results and Discussion

weight was determined as 4 million by pullulan (Wako Pure

4.1 Measurement of MW using HPSEC

Chemicals, Chiba, Japan). The flow rate was 0.8 mL/min, and

MW of purified sample was measured using HPSEC (Fig.

the eluate was monitored with an RI detector (2410 RI Detector,

2). Chromatograph of RI showed a pattern that peaks at RT of

Waters), and the column temperature was maintained at 40°C.

10.6 min and showed ACE inhibitory activity. This top MW peak

3.5 Detection of protein, carbohydrate, and uronic acidcorresponded to 3.0 kDa on the calibration curve. So, purified

The detection of protein was carried out in accordance withninhydrin reaction. The purified sample was dissolved as 5.0 mg/

sample that exhibited ACE inhibitory activity has a molecularweight of about 3.0 kDa.

mL in 5.0% acetonitrile solution with 0.1% TFA and spotted ona thin-layer chromatography plate (Silica gel 60 F254, 5.0 cm ×10.0 cm, Merck, Darmstadt, Germany) and 0.1% glutamic acid
3.0kDa	was used as a positive control. The plate was developed for 25
min in butanol: glacial acetic acid: distilled water (2:1:2, v/v),
and dried for 5 min and the samples were visualized with 0.2%
ninhydrin solution.
The detection of carbohydrate was carried out in according
using the phenol-sulfuric acid method modified by Hodge et al.22
Purified sample (500 mg) was dissolved in 1.0 mL of 0.05 M
phosphate buffer (pH 7.0). 1.0 mL of the solution was dispensed
in a test tube, followed by addition of 1.0 mL of 5.0% phenol
solution and 5.0 mL of concentrated sulfuric acid, and incubation

Fig. 2High pressure size exclusion chromatography (HPSEC)

at 27°C for 15 min. Absorbance was measured at 485 nm withchromatogram of fractions. The purified fraction withthe spectrophotometer (UV-120, Shimadzu, Kyoto, Japan), using

ACE inhibitory activity was applied to a Shodex-

distilled water as the blank.

Ohpak SB-803HQ column with 0.1 M NaNO3 for 20

Presence of uronic acid in the sample was detected using

min at a flow rate of 0.8 mL/min. The line shows thethe meta-hydroxydiphenyl method23. The purified sample (200chromatogram (relative intensities at the RI detector).

mg) was dissolved in 400 μL of distilled water and shaken with

The molecular weight of the highest peak (ca. 10.6 min)2400 μL of 18 M sulfuric acid/sodium tetraborate in a vortex

was calculated as 3.0 kDa from the standard curve.

mixer, followed by heating at 100°C for 5 min. After cooling,
40 μL of 0.15% meta-hydroxydiphenyl in 0.5% NaOH wasadded, shaken, and incubated at 27°C for 5 min. Absorbance wasmeasured at 520 nm.
3.6 Structural analysis by nuclear magnetic resonance (NMR)

4.2 IC50 value of ACE inhibitory activity of purified sample
IC50 value of ACE inhibitory activity of purified sample was
0.36 mg/mL, which was lower than that measured at Step 1 (2.57

The purified sample, sucrose, glucose, and cellulose were

mg/mL). This IC50 level was comparable with that reported for

dissolved in deuterium oxide solution (20 mg/0.6 mL). The

hot water of mushroom (Hiratake: 0.65 mg/mL, Hatakeshimeji:

entire sample volume of 0.6 mL was transferred to 5.0 mm NMR

0.76 mg/mL10 and enzymatic hydrolysate of fish meat (0.65 ~sample tube (BMS-005B, Shigemi Inc., Tokyo, Japan).

3.24 mg protein/mL12.

NMR measurement was performed using Varian NMR

4.3 Detection of proteins, carbohydrates, and uronic acid4

Purified sample was tested with the ninhydrin reaction

3. The resonances of purified sample linked to hemiacetal

to detect proteins. Glutamic acid, used as a positive control,hydrogen in the region of δ4.6 ~ 5.4 ppm and hydrogen boundshowed orange color after the reaction, while the purified sample

to oxy carbon (COH) in the region of δ3.4 ~ 4.6 ppm. Therefore,

did not. Thus, the purified sample did not contain peptide or

the sample may consist of mainly carbohydrates. The lines

amino acid. Furthermore, the purified sample developed orange

broadening the spectrum indicate existence of iteration of similarcolor on subjecting it to the phenol-sulfuric acid method tocarbohydrates (main chain: arrowed line) and other carbohydrate

detect carbohydrates. Thus, the purified sample may containbackbone (minor structure). 13C NMR spectra of purified sample

carbohydrates. Additionally, the purified sample was tested using

showed resonance that proceeds to carboxyl in the region of

the meta-hydroxydiphenyl method, and the concentration of

δ170 ~ 175 ppm. Thus, the purified sample may contain aciduronic acid in the purified sample was calculated as 171.0 μg/

carbohydrate (Fig. 4). These results indicate that this component

mL.

must have iteration of acid carbohydrates.

4.4 1H and 13C NMR spectroscopy of lyophilized sample of ACE

1inhibitor purified fraction
1

H NMR spectra of pectin or de-esterified pectin24 werecompared with that of polygalacturonic acid (poly-GalA) as
the main component of pectin25. The main chain resonances of

H NMR spectra of the purified sample are shown in Fig.

HDO	-COCH3	
 -CH3	

6

Fig. 3

1

5

4

0

1

2

3

ppm	

H NMR spectrum of the fraction with angiotensin I-converting enzyme (ACE) inhibitory activity (deuterium water, 25°C). The

resonances of purified sample linked to hemiacetal hydrogen in the region of δ4.6 ~ 5.4 ppm and hydrogen bound to oxy carbon(COH) in the region of δ3.4 ~ 4.6 ppm. Therefore, the sample may consist of mainly carbohydrates. The lines broadening thespectra indicate existence of iteration of similar carbohydrates (main chain: arrowed line) and other carbohydrate backbone(minor structure).

>C=O	

180	

Fig. 4

160	

140	

120	

100	

80	

60	

40	

20	

ppm	

13

C NMR spectrum of the fraction with angiotensin I-converting enzyme (ACE) inhibitory activity (deuterium water, 25°C).

13

C NMR spectra of purified sample showed resonance that proceeds to carboxyl in the region of δ170 ~ 175 ppm.

5

purified sample (arrowed line) agree with those of pectin, de-H1	esterified pectin, and poly-GalA, but not with the resonance

Main Chain	

of methoxy group (-OCH3). Thus, the main chain of purified

Group A	

sample was suggested to be polymer of GalA by α-1,4 linkage

H4	

H3	

H4	

H1	

H5	

H4	

H1	
 H5	

Group C	H NMR spectra showed that the signal proceeds to

H5	
 H4	

H1	

ppm	
3.6	
3.8	
4.0	
4.2	
4.4	
4.6	
4.8	
5.0	
5.2	
5.4	

main and minor structure. 1H and 13C NMR and measurement
results of COSY, HMQC, and NOESY are shown in Table 1.
Minor structure also gave the other signals, and COSY spectra,
depending on the minor structure (Fig. 5), showed the correlation
among three hexose skeletons (Group A ~ C). 13C NMR spectra
showed the existence of carbonyl group in the C6 position, so
the purified sample may contain uronic acids, such as glucuronic

H2	
H3	

H2	

H3	
 H2	

Group B	

like pectin acid.
1

H5	

H3	
 H2	

Group A	Group B	Group C	
5.4	
 5.2	
 5.0	
 4.8	
 4.6	
 4.4	
 4.2	
 4.0	
 3.8	
 3.6	
 3.4	
 ppm	

acid and mannuronic acid.

Fig. 5

Correlation spectroscopy (COSY) spectra of theProtons on C1 ~ C2 and C2 ~ C3 of group A have afraction with angiotensin I-converting enzyme (ACE)

J-coupling with frequency of 8 Hz and 10 Hz, so the proton

inhibitory activity (deuterium water, 25°C). COSY

conformation on C1 ~ C2 and C2 ~ C3 is suggested to be diaxial

spectra showed the correlation among three hexose
skeletons (Group A ~ C).

1
13
Table 1 Attribution of H and C spectra.

No.

δ(ppm)*1

Multiplicity*2

J (Hz)

13
C
No.

Group

Atribution

1

3.5

dd

10，8

25

A

β－GalA*3 H2，C2

2

3.71

dd

11，4

19

B

α－GalA3

3.7-3.8m―

19Main chain

27

A

1

H

H2，C2

α－GalA H2，C2β－GalA

H3，C3

4

3.81

S

―

―

―

5

3.83

dd

11，4

19

C

α－GalA

H2，C2

6

3.91

dd

11，4

21

B

α－GalA

H3，C3

7

4

m

―

20

Main chain ，C

α－GalA

H3，C3

8

4.09

bs

―

28

A

β－GalA H5，C5

9

4.28

dd

4，2

23

B

α－GalA

10

4.41

d

3

29

A

β－GalA H4，C4

11

4.4

m

―

30

Main chain ，C

α－GalA H4，C4

12

4.46

bs

―

22

C

α－GalA

H5，C5

13

4.6

d

8

32

A

β－GalA

H1，H1

14

4.82

bs

―

24

Main chain

α－GalA

H5，C5

15

4.85

bs

―

26

B

α－GalA

H5，C5

16

5.05

d

4

33

B

α－GalA

H1，C1

17

5.1

m

―

33

Main chain

α－GalA

H1，C1

18

5.31

d

4

31

C

α－GalA

H1，C1

―

―

―

―

34

―

－COOHCH3

―

―

―

―

35

A

β－GalA

―

―

―

―

36

Main chain ，C

α－GalA C6

dd (dual-double line),

―

―

―

―

37

B

α－GalA C6m (multiple line),－COOCH3

H4，C4*1

Signal of HDO was δ4.77 ppm.

*2

Multiplicity: s (single line),C6d (double line),bs (wide range of single line)*3

GalA: Galacturonic acid6

position. The proton on C4 shows doublet with narrow splitting

Table 2

Area intensity of main chain and minor structure(1H spectrum).

width, and the proton on C5 has no splitting. Therefore, theproton on C3 ~ 4 and C4 ~ C5 must have a J-coupling with a

SignalArea intensity
ratio

conformation on C3 is axial, but C4 proton conformation could

Main Chain H1 ＋ Group B H1

6.8

not be determined. Group B has protons on C1 ~ C2, C2 ~ C3

Main Chain H3 ＋ Group C H3

5.8

frequency of under 3 Hz. These results indicate that the proton

and C3 ~ C4, of which a J-coupling showed frequencies of 4

Group A

H1

1

Hz, 11 Hz, and 4 Hz, respectively. Each proton conformation

Group A

H2

1

must be equatorial, axial, and equatorial and axial position.

Group B

H3

2.1

However, among group C, the proton conformation on C1 andGroup BH4

1.9

C2 may be equatorial and axial position, similar to that on group

Group C

H1

0.6

B. The protons on C3 and C4 present overlapped signal derived
other protons, rendering it difficult to calculate their J-coupling
frequencies. Therefore, the proton on C3 is suggested to be inan axial position with J-coupling between that on C2, but theblock to be main chain: group A: group B: group C = 8 : 2 : 3

conformation of the proton on C4 could not been assigned.

: 1. The results indicate that the purified sample may consist of

These conformations suggest that both protons on C2 and C4

mainly GalA linked each other with α-1,4-bond (partially β-1,4of group A and B are equatorial and axial position, and theirlinkage). Comparison of integrated values of the signal for theminor component is GalA, similar to that in the main chain.

structures of terminal and main chain indicated that the purified

Glucuronic acid and mannuronic acid are excluded as theirsample should have less than 10 uronic acid moieties. Thus,

components because the protons on C2 and C4 on the former are

ACE inhibitor substance in tomato juice is speculated to have ain axial position and the protons on C2 and C4 on the latter aremolecular weight of about 3.0 kDa with less than 10 uronic acidin equatorial and axial position.

moieties.

The proton conformation on C2 of group C is in axial
position, but that on C4 is unclear. The component might be

5 Conclusions

GalA or glucuronic acid. Based on the proton conformation on

IC 50 level of the purified sample showed lower ACE

C1 of group A, both groups B and C should have β-structure

inhibitory activity compared to those of the enzymaticand α-structure, respectively. GalA in main chain, of whichhydrolysate of fish meat 12 and the hot water extract ofthe proton position on C1 is located next to group B, and themushroom17. The present study indicates the fraction containing

partially confirmable microstructure has J-coupling frequency

about 3 kDa of uronic acid-derivatives from the tomato juice hasof 4 Hz, is considered as the α-structure. Thus, the main chain

ACE inhibitory activity.

may consist of GalA, which binds each other with α1,4-linkage.
The signals of the minor structure are more definitive thanAcknowledgmentsthat of the main chain. Therefore, these minor carbohydrates

We thank Ms. Chiyo Sano for supporting this work, andshould be located in the sugar chain terminal. HMBC spectraDrs. Y. Nakano, A. Wadano, R. Sakamoto of Osaka Prefecture

showed that the proton on C1 of group A did not correlate with

University, for excellent advice and guidance. A part of thisanother carbohydrate, but the proton on its C4 correlated withstudy was supported by a grant from KAGOME CO., Ltd..

carbon on C1 of group B or the main chain. The protons on C1
of group B correlate with carbon on C4 of group A and main
chain, while the proton on C4 did not correlate with another

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